Fluorescent nanoparticles (FNP) are becoming increasingly popular in biomedical imaging and tagging. Compared to molecule-based fluorophores, FNP are typically brighter and more photostable. They can be functionalized with more than one tagging molecule, used for the tagging and tracing of specific molecules, cells, tissues. Brighter fluorescence facilitates more sensitive labeling, and in many cases, a lower limit of detection. In flow cytometry it may open new limits of detecting low number of receptors on cells and exosomes. NanoScience Solutions LLC (NSS) is developing exceptionally bright nanoporous silica FNP (suggested commercial name ?Star-dots?), nanoporous silica FNP, in which fluorescent dye molecules are physically encapsulated in a specific nano-environment of cylindrical nanochannels. To the best of our knowledge, Star-dots are the brightest currently available FNP (vetted by multiple PI?s publications in high-level journals and numerous citations). Star-dots exhibit good colloidal stability (months/years); no dye leakage; no fast aggregation and negligible spectral change in different media; no blinking; low photobleaching compared to free (non-encapsulated) dye; and the absence of non-fluorescent particles. Due to close proximity of encapsulated dye molecules to each other, which allows the attainment of quantum coupling between dye molecules, development of particles with fluorescent spectra of almost arbitrary complexity is feasible; including multiple spectra that can be excited with the same wavelength. Such particles are of big interest to various multiplexed assays, in particular, to multi-spectral flow cytometry. Physical encapsulation (versus the currently used standard chemical bonding) of existing fluorescent dyes allows the use of a large number of commercially available dyes with no modification, including hard-to-modify near-infrared dyes. However, despite these advantages, Star-dots cannot be used for biomedical applications as of yet. The critical missing part is functionalization of Star-dots with tagging molecules. Standard ways of silica functionalization kill fluorescence of Star-dots. This STTR Phase I project will 1) address this critical problem and 2) test the feasibility of using Star dots for multiplexing applications. Specifically, we propose: 1) Demonstrate that feasibility of functionalizing Star-dots with functional (tagging) molecules while preserving Star-dot high fluorescent brightness. Coating Star-dots with protective molecules (like polyethylene glycol) to decrease nonspecific interactions will also be explored. 2) Test Star dots for multiplexing applications. Our preliminary data demonstrate the development of multiple fluorescent spectra excited with one wavelength, but only when using micron size particles. Scaling down the size of these particles to nanoscale is highly non-trivial and always associated with high risk. The academic partner will deal with all characterizations of Star-dots. The small business partner has the experience of development of various Star-dots. The successful completion of Phase I will substantially decrease risk associated with the ambitious goal of introducing Star dots into the biomedical arena. Creation of new, bright, stable, non-toxic fluorescent markers, which, in many respects, are superior to the existing fluorescent nanoparticles, will provide a new tool to the biomedical community and facilitate research involving detection, transport, flow-cytometry and the imaging of biochemical events within living cells, tissues. It will lay the groundwork for the development of commercial prototype products (Phase II). One of the expected specific goals of Phase II will be the development of Star-dots for flow cytometry together with our partners (such as Beckman Coulter, one of the leaders in flow cytometry ? see their letter of interest).